Alzheimer's disease (AD) is a common dementing brain disorder of the elderly. The key features of the disease include progressive memory impairment, loss of language and visuospatial skills, and behavior deficits. These changes in cognitive function are the result of degeneration of neurons in the cerebral cortex, hippocampus, basal forebrain, and other regions of the brain. Neuropathological analyses of postmortem Alzheimer's diseased brains consistently reveal the presence of large numbers of neurofibrillary tangles in degenerated neurons and neuritic plaques in the extracellular space and in the walls of the cerebral microvasculature. The neurofibrillary tangles are composed of bundles of paired helical filaments containing hyperphosphorylated tau protein (Lee, V. M and Trojanowski, J. Q, The disordered Cytoskeleton in Alzheimer's disease, Curr. Opin. Neurobiol. 2:653 (1992)). The neuritic plaques consist of deposits of proteinaceous material surrounding an amyloid core (Selkoe, D. J., "Normal and abnormal biology of the .beta.-amyloid precursor protein", Annu. Rev. Neurosci. 17:489-517 (1994)).
Evidence suggests that deposition of amyloid-.beta. peptide (A.beta.) plays a significant role in the etiology of Alzheimer's disease. A portion of this evidence is based upon studies which have been generated from data with regard to familial Alzheimer's disease. To date, this aggressive form of Alzheimer's disease has been shown to be caused by missense mutations in (at least) three genes: the amyloid precursor protein (APP) gene itself (Goate, A. et al., "Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease", Nature 349:704-706 (1991) and Mullan, M. et al., "A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of .beta.-amyloid", Nature Genet. 1:345-347 (1992)), and two genes termed presenilins 1 and 2 (Sherrington, R. et al., "Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease", Nature 375:754-760 (1995) and Rogaev, E. I. et al., "Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene", Nature 376:775-778 (1995)). The missense mutations in APP are located in the region of the protein where proteolytic cleavage normally occurs (see below), and expression of at least some of these mutants results in increased production of A.beta. (Citron, M. et al., "Mutation of the .beta.-amyloid precursor protein in familial Alzheimer's disease increases .beta.-amyloid production", Nature 360:672-674 (1992), Cai, X-D. et al., "Release of excess amyloid.beta. protein from a mutant amyloid.beta. protein precursor", Science 259:514-516 (1993) and Reaume, A. G. et al., "Enhanced amyloidogenic processing of the beta-amyloid precursor protein in gene-targeted mice bearing the Swedish familial Alzheimer's disease mutations and a humanized A.beta. sequence", J. Biol. Chem. 271:23380-23388 (1996)). Initial analyses of the structure of the presenilins have not suggested whether or not they might alter production of A.beta., however, recent data has indicated that these mutations cause an increase in A.beta. secretion (Martins, R. N. et al., "High levels of amyloid-.beta. protein from S182 (Glu.sup.246) familial Alzheimer's cells", 7:217-220 (1995) and Scheuner, D. et al., "Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease", Nature Medicine 2:864-870 (1996)). Thus, increased production of A.beta. is associated with Alzheimer's disease. Corroborating evidence has been derived from at least two other sources. The first is that transgenic mice which express altered APP genes exhibit neuritic plaques and age-dependent memory deficits (Games, D. et al., "Alzheimer-type neuropathology in transgenic mice overexpressing V717F .beta.-amyloid precursor protein", Nature 373:523-525 (1995); Masliah, E. et al., "Comparison of neurodegenerative pathology in transgenic mice overexpressing V717F .beta.-amyloid precursor protein and Alzheimer's disease", J. Neurosci. 16:5795-5811 (1996); Hsiao, K. et al., "Correlative memory deficits, A.beta. elevation, and amyloid plaques in transgenic mice", Science 274:99-103 (1996)). The second piece of evidence comes from study of patients suffering from Down's syndrome, who develop amyloid plaques and other symptoms of Alzheimer's disease at an early age (Mann, D. M. A. and M. M. Esiri, "The pattern of acquisition of plaques and tangles in the brains of patients under 50 years of age with Down's syndrome", J. Neuro Sci. 89:169-179 (1989)). Because the APP gene is found on chromosome 21, it has been hypothesized that the increased gene dosage which results from the extra copy of this chromosome accounts for the early appearance of amyloid plaques (Kang, J. et al., "The precursor protein of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor", Nature 325:733-736 (1987); Tanzi, R. E. et al., "Amyloid .beta. protein gene: cDNA, mRNA distribution and genetic linkage near the Alzheimer locus", Science 235:880-884 (1987)). Taken together with the evidence derived from cases of familial Alzheimer's disease, the current data suggests that genetic alterations which result in an increase in A.beta. production can induce Alzheimer's disease. Accordingly, since A.beta. deposition is an early and invariant event in Alzheimer's disease, it is believed that treatment which reduces production of A.beta. will be useful in the treatment of this disease.
The principal component of the senile plaque is the 4 kDa .beta.-amyloid peptide (A.beta.). Ranging between 39 and 43 amino acids in length, A.beta. is formed by endoproteolysis of APP. Alternative splicing generates several different isoforms of APP; in neurons, the predominant isoform is of 695 amino acids in length (APP695). As APP traverses the endoplasmic reticulum (ER) and trans-Golgi network (TGN), it becomes N- and O-glycosylated and tyrosine-sulfated. Mature holoprotein can be catabolized in several compartments to produce both non- and amyloidogenic APP fragments.
APP is expressed and constitutively catabolized in most cells. The dominant catabolic pathway appears to be cleavage of APP within the A.beta. sequence by an enzyme provisionally termed .alpha.-secretase, leading to release of a soluble ectodomain fragment known as APPs.alpha.. In contrast to this non-amyloidogenic pathway, APP can also be cleaved by unidentified enzymes known as .beta.- and .gamma.-secretase at the N- and C-termini of the A.beta., respectively, followed by release of A.beta. into the extracellular space. Several different C-terminal fragments are produced as intermediates in APP catabolic processing; of particular interest is the production of an intracellular an 12 kDa C-terminal fragment (C100) which is produced following .beta.-secretase activity and contains the entire A.beta. sequence.
Abundant evidence suggests that extracellular accumulation and deposition of A .beta. is a central event in the etiology of AD. However, recent studies have also proposed that increased intracellular accumulation of A.beta. or amyloid containing C-terminal fragments may play a role in the pathophysiology of AD. For example, overexpression of APP harboring mutations which cause familial AD results in the increased intracellular accumulation of C100 in neuronal cultures and A.beta..sub.42 in HEK 293 cells. Moreover, evidence suggests that intra- and extracellular A.beta. are formed in distinct cellular pools in hippocampal neurons and that a common feature associated with two types of familial AD mutations in APP (`Swedish` and `London`) is an increased intracellular accumulation of A.beta..sub.42. Thus, based on these studies and earlier reports implicating extracellular A.beta. accumulation in AD pathology, it appears that altered APP catabolism may be involved in disease progression.
Numerous studies have suggested that proteolytic cleavage of APP occurs within acidic compartments of the cell based on the inhibitory actions of agents which are known to disrupt intracellular pH and/or acidic organelles. For example, exposure of cells to the monovalent ionophore, monensin, or high concentrations of ammonium chloride (NH.sub.4 Cl) been shown to decrease APP proteolytic processing accompanied by concomitant alterations in full-length cellular APP. Similarly, the vacuolar H.sup.+ -ATPase inhibitor bafilomycin A1 (baf A1) has been reported to produce alterations in APP catabolism which are both cell-type and APP mutation specific. However, the ionophores noted above have been found to be toxic, unacceptably inhibit ATP formation, and alter cellular viability and endosomal and lysosomal function.
Thus, in view of the anticipated benefits of modulating APP catabolism as a treatment for diseases such as AD, compositions and methods for modulating APP catabolism in APP-containing cells which do not substantially alter the viability of those cells, have been desired and are addressed by the present invention.